Effects of increased anionic charge in the beta-globin chain on assembly of hemoglobin in vitro.
Studies on assembly in vitro of alpha-globin chains with recombinant beta16 Gly-->Asp, beta95 Lys-->Glu, beta120 Lys-->Glu and beta16 Gly-->Asp, 120 Lys-->Glu human beta-globin chain variants in addition to human betaA- and betaS-globin chains were performed to evaluate effects of increased anionic charge in the beta chain on hemoglobin assembly using soluble recombinant beta-globin chains expressed in bacteria. A beta112 Cys-->Asp change was also engineered to monitor effects on assembly of increased negative charge at alpha1beta1 interaction sites. Order of tetramer formation in vitro under limiting alpha-globin chain conditions showed Hb betaG16D, K120E = Hb betaK120E = Hb betaK95E > Hb betaG16D > Hb A > Hb S >>> Hb betaC112D. In addition, beta112 Cys-->Asp chains exist as monomers rather than beta4 tetramers in the absence of alpha chains, and the beta chain in Hb betaC112D tetramers was readily exchanged by addition of betas. These results suggest that affinity between alpha and beta chains is promoted by negatively-charged beta chains up to a maximum of two additional net negative charges and is independent of location on the surface except at the alpha1beta1 interaction site. In addition, our findings show that beta112 Cys on the G helix is critical for facilitating formation of stable alphabeta dimers, which then form functional hemoglobin tetramers, and that beta112 Cys-->Asp inhibits formation of stable alpha1beta1 and beta1beta2 interactions in alpha2beta2 and beta4 tetramers, respectively.
Adachi
K
K
Division of Hematology, The Children's Hospital of Philadelphia, and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Yamaguchi
T
T
Pang
J
J
Surrey
S
S
D000838:Anions; D005914:Globins / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D055672:Static Electricity
D000838:Anions; D006454:Hemoglobins; D011994:Recombinant Proteins; D005914:Globins; D010100:Oxygen